BaculoELISA Titer Kit User Manual

User Manual Cat. No. 631412 PT3953-1 (PR752243) Published 5 May 2007

Table of Contents I. Introduction...3 II. III. IV. List of Components...4 Additional Materials Required...5 BaculoELISA Titer Kit Protocol Overview...6 V. BaculoELISA Titer Kit Protocol...7 A. Protocol: Seeding Insect Cells... 7 B. Protocol: Preparing Serial Virus Dilutions... 7 C. Protocol: Infecting Insect Cells... 8 D. Protocol: Preparing Solutions... 8 E. Protocol: Preparing the ELISA Plate... 8 F. Protocol: Performing the ELISA... 8 G. Protocol: Calculating Baculovirus Titers... 9 VI. Troubleshooting... 11 VII. References... 11 List of Figures Figure 1. The BaculoELISA Titer Kit protocol... 6 Figure Data required to estimate the viral titer of a test virus... 9 List of Tables Table I: Example Series Dilution Panel... 7 Table II: Troubleshooting the BaculoELISA Titer Kit... 11 Notice to Purchaser Clontech products are to be used for research purposes only. They may not be used for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. Clontech products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories, Inc. Biohit and epet are trademarks of Biohit. Corning and Costar are registered trademarks of Corning Inc. Drummond is a trademark of Drummond Scientific Company. Microsoft and Excel are registered trademarks of Microsoft Corporation. Parafilm is a registered trademark of American National Can Company Sigma-Aldrich is a trademark of Sigma-Aldrich Biotechnology L.P. Clontech, Clontech logo and all other trademarks are the property of Clontech Laboratories, Inc. unless noted otherwise. Clontech is a Takara Bio Company. 2007 Protocol No. PT3953-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR752243 A Takara Bio Company

I. Introduction BaculoELISA Titer Kit Clontech s BaculoELISA Titer Kit enables you to determine baculovirus titers based on the expression of p35, an early-expressed viral protein that prevents cellular apoptosis and is essential for infectivity. p35 is initially detected in the host cytosol 4 12 hours after infection, before morphological changes or virus budding are observed (Gershburg et al, 1997; Hershberger et al, 1994). The BaculoELISA Titer Kit accurately measures the ability of the virus to infect cells and express virally-encoded proteins. Since the goal of the baculovirus system is to express recombinant proteins, this is an extremely relevant assay with straightforward results. The kit allows you to determine the titer of virus stocks in infectious units per ml (IFU/ml) or plaque forming units per ml (PFU/ml), convenient parameters for optimizing infection kinetics, by comparing the p35 levels produced by your virus(es) of interest with the p35 levels produced by a virus of known titer. Assay results can be obtained in two days, with approximately 3 4 hours of hands-on time. This kit can be used to titer any AcMNPV-based baculovirus, including all commercially available baculovirus systems. Advantages of the baculovirus system The baculovirus expression system is widely used to overexpress recombinant proteins for several reasons: Baculoviruses expressing recombinant proteins of interest can be generated and amplified easily in insect cells. Insect host cells produce large amounts of recombinant protein. The recombinant protein expressed in insect cells is usually similar in structure, biological activity, and immunological reactivity to the naturally occurring protein, because insect host cells and mammalian cells perform similar post-translational processing. Baculovirus expression is an excellent choice for toxic and/or secreted proteins, and for proteins that are too large or complex to be expressed in bacteria. Scaling up recombinant protein production is simple because insect cells are easy to culture as adherent, spinning or shaking cultures. The importance of titering baculovirus stocks Robust, reproducible protein expression begins with an accurate titer determination for the target virus. Accurate titration allows you to precisely control the concentration of virus used to infect your insect cells. Furthermore, titration ensures that virus has been produced, and helps to verify proper cell growth and virus amplification. The timing and level of expression of your target protein is highly dependent on the ratio of infectious virus particles to insect cells, or multiplicity of infection (MOI). Typically, the first step to optimize the expression of a new target protein is to perform a time course of infection, by infecting small numbers of cells with a range of MOIs (e.g., 2 50 virus particles per cell), and harvesting samples at several time points between 36 72 hours postinfection. The best combination of MOI and time of infection can then be used to express the target protein in large quantities. If the MOI is too low, too few cells will be infected and the infection will not be well synchronized. If the MOI is too high, the metabolic strain will reduce the level of expression and/or the correct processing of the protein of interest. The best results are usually obtained with infections using an MOI of ~5 20. This ensures that all cells are infected simultaneously, and that multiple virus particles infect each cell, for strong expression of your target protein. Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3953 1 A Takara Bio Company Version No. PR752243

II. List of Components Store ELISA plates and 20% Tween at room temperature. Store the control virus at 4 C, protected from light. Store all other kit components at 4 C. The BaculoELISA Titer Kit contains sufficient reagents for up to 10 baculovirus titrations. BaculoELISA Titer Kit (Cat. No. 631412) 200 µl Control Virus 230 µl Capture Antibody 15 µl Detection Antibody 100 µl Avidin-HRP Conjugate 3 ml 2X J-RIPA Buffer 60 ml 10X Tris-Buffered Saline (TBS) 5 ml 20% Tween 11 ml TMB Substrate Reagent A 11 ml TMB Substrate Reagent B 2 96-Well ELISA Plates Supporting Documents BaculoELISA Titer Kit User Manual (PT3953-1) BaculoELISA Titer Kit Protocol-at-a-Glance (PT3953-2) Visit our web site at www.clontech.com for a current list of Baculovirus Expression products. Protocol No. PT3953-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR752243 A Takara Bio Company

III. Additional Materials Required The following materials are required but not supplied: Sf9 or Sf21 insect cells We recommend IPLB-Sf21 Insect Cells (Cat. No. 631411) Insect cell medium We recommend BacPAK Complete Medium (Cat. No. 631403) or TNM-FH Medium Hemacytometer Trypan blue solution, to measure cell viability Multichannel pipettor (20 200 µl) Reservoir trays 96-Well tissue culture plates Cell culture incubator (27 C; humidified by water) Microplate shaker Microplate reader for either 600 700 nm (maximum absorbance at ~650 nm) or 430 470 nm (maximum absorbance at ~450 nm) To measure absorbance at 430 470 nm only: acid solution (e.g., 2 N HCl) to stop the reaction Materials to perform the multiple wash and reagent removal steps required for all standard ELISA protocols: Recommended method: Use a multichannel repeating pipettor with 1 1.2 ml tips, and a vacuum aspirator to add and remove liquids from the wells, respectively. Multichannel repeating pipettor and tips: We recommend the Biohit epet multichannel pipettor (Biohit Cat. Nos. 71081XET, 710812ET, 71080XET, or 710802ET). Multichannel vacuum aspirator: Eight-channel vacuum aspirators compatible with 96-well plates are available from several companies, including Corning (Costar Vacuum Aspirator, Cat. No. 4930) Drummond (3-000-093), Sigma (Cat. No. M2656-1EA), and Inotech (Cat. No. IV-503). Vacuum trap systems that can use eight-channel vacuum adaptors are available from Corning (Cat. No. 4931) and Inotech. Vacuum adaptors can also be purchased alone and inserted into proper-size tubing connected to a vacuum trap flask. A complete system, including a vacuum trap bottle, is available from Inotech (Cat. No. IV-500). Alternative Method I Squeeze Bottle: Use a squeeze bottle to dispense wash buffer. Remove liquids by inverting and shaking out the plate, then remove additional liquid by tapping on a clean, dry paper towel. Squeeze bottle Alternative Method II ELISA Plate Washer: ELISA plate washers provide a very convenient method to both add wash buffer and to aspirate wash buffer and reagents from ELISA plates. However, the use of an ELISA plate washer will require more TBS-T wash buffer than the amount provided in the kit. You will need to make additional TBS-T, according to the recipe in Part V.D. ELISA plate washer Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3953 1 A Takara Bio Company Version No. PR752243

IV. BaculoELISA Titer Kit Protocol Overview Please read the entire protocol before starting. Successfully implementing the BaculoELISA Titer Kit protocol requires performing the steps described below, all of which are included in this user manual. The BaculoELISA Titer Kit follows a two-day protocol which includes seeding insect cells, diluting viral stocks or samples, infecting cells, and performing the ELISA (Figure 1). On the first day, you will seed insect cells (Sf9 or Sf21) in a 96-well tissue culture plate, allow them to settle, and infect them with serial dilutions of the control virus and your viruses of unknown titer (~1.5 hr). Separately, you will add the capture antibody to the 96-well ELISA plate (~20 min) and store it overnight at 4 C. On the second day, you will lyse the infected cells and measure p35 levels by ELISA (3-4 hours). Titers can be calculated manually, or using the workbook provided on our website, www.clontech.com. Attention Attention: Please note that, for this assay, the control virus not only serves as a positive control, but also as a reference for the purpose of calculating the titer of your test viruses. For reagent preparation, please be aware that this protocol is written based on the total number of viruses being analyzed (n), including the reference virus. Control virus Serial dilutions of known and unknown viruses Unknown (target) viruses A 650 Virus concentration Lyse and transfer to immunoassay plate Add virus dilutions to insect cells (fresh 96-well plate) Figure 1. The BaculoELISA Titer Kit protocol. Make serial dilutions of the control (reference) virus of known titer (included with the kit) and of your viruses of unknown titer, and infect insect cells. Incubate for 24 hr, then lyse and transfer the lysate to the prepared ELISA plate. Perform the ELISA and measure absorbance at 650 nm. Use the standard curve generated from the reference virus to determine the titers of your test baculoviruses. The BaculoELISA Titer Kit contains sufficient reagents for up to 10 baculovirus titrations. The titrations can be performed all in one ELISA plate, or in two separate plates, on separate occasions: If you perform all the assays at one time (using a single plate), you can titer up to 11 test viruses and the reference virus, or up to 10 test viruses with two repeats of the reference virus. Alternatively, you can perform two separate assay procedures and titer a total of 10 test viruses, plus one repeat of the reference virus on each plate. Protocol No. PT3953-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR752243 A Takara Bio Company 6

V. BaculoELISA Titer Kit Protocol Day 1 (V.A V.E) 1½ hr Attention A. Protocol: Seeding Insect Cells 1. Collect cultured Sf9 or Sf21 insect cells (e.g., from a spinner flask or plate), and count viable cells using a hemacytometer and trypan blue. Dilute in insect medium to 1 x 10 6 cells/ml. Note: We recommend using IPLB-Sf21 Insect Cells (Cat. No. 631411) and either BacPAK Complete Medium (Cat. No. 631403) or TNM-FH Medium. For more detailed information about culturing and counting insect cells and about handling baculoviruses, please refer to the BacPAK Baculovirus Expression System User Manual (PT1260-1). Seed 100 µl of insect cell suspension (10 5 cells/well) into each well of n columns of a 96-well culture plate, where n = the total number of viruses, including the reference virus. Note: The amount of p35 in each well will depend on the viral titer, and to a lesser extent, on the number of cells in the well. Therefore, it is important to have similar numbers of cells in each well. Let the cells attach for at least 15 min at room temperature. They should be ~90% confluent. Alternatively, seed 7 x 10 4 cells/well and incubate overnight at 27 C in an incubator, until they reach ~90% confluency. Note: Excluding serum or adding Pluronic F68 will greatly extend the time required for cell attachment. B. Protocol: Preparing Serial Virus Dilutions For each of n columns of a microtiter plate: Attention 1. 4. Add 100 µl of insect cell medium to each well. Add 50 µl of the first baculovirus sample to the first well of the column (row A). Mix well by gently pipetting up and down several times. Repeat, one column per baculovirus. Note: Baculoviruses can be destroyed by UV light. Turn off fluorescent lights while handling the baculoviruses. Using a multichannel pipette, transfer 50 µl from the first well of the column to the second well, mix thoroughly by pipetting, and transfer 50 µl of the suspension to the next well. Repeat until the serial dilution is completed. Repeat steps 2 3, one column per virus. Table I: Example Series Dilution Panel 1 2 3 4 5 6 7 8 9 10 11 12 A Reference 1:3 Test 1:3 B Reference 1:9 Test 1:9 C Reference 1:27 Test 1:27 D Reference 1:81 Test 1:81 E Reference 1:243 Test 1:243 F Reference 1:729 Test 1:729 G Reference 1:2187 Test 1:2187 H Reference 1:6561 Test 1:6561 Table I: Example of a series dilution panel for the reference virus (Column 1) and one unknown test virus (Column 2). Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3953 1 A Takara Bio Company Version No. PR752243

V. BaculoELISA Titer Kit Protocol continued C. Protocol: Infecting Insect Cells 1. Label the insect cell culture plate (Protocol A) with the dilutions corresponding to each well of the dilution panel (Protocol B). Gently overlay 50 µl of the diluted virus stocks on the corresponding wells containing the insect cells. If you start with the most dilute virus stocks (row H), you can do the overlays without changing pipet tips. Incubate in a cell culture incubator (27 C; humidified by water) for 16 20 hr. Attention Note: Baculoviruses can be destroyed by UV light. Make sure the infected plates are not exposed to light. D. Protocol: Preparing Solutions To perform the entire assay procedure in one plate, prepare the following. Dilute 10X TBS to 1X TBS 1.5 ml 10X TBS 9.5 ml H 2 O Final volume: 11 ml 1X TBS Recipes Prepare TBS-Tween (TBS-T; final concentration 0.05% Tween by volume) 50 ml 10X TBS 450 ml H 2 O 1.25 ml 20% Tween Final volume: 0.5 L TBS-T Note: If you plan to perform the assay on two separate occasions, you will need to scale down the solutions: Prepare n ml 1X TBS where n = the total number of viruses. Prepare n x 45 ml TBS-T where n = the total number of viruses. E. Protocol: Preparing the ELISA Plate 1. Dilute n x 19 µl of the capture antibody stock in n x 900 µl TBS without detergent. (e.g., for 12 viruses, dilute 228 µl of capture antibody in 10.8 ml of TBS.) Add 100 μl of diluted capture antibody to each well of the columns in the ELISA plate that you will use. Cover the ELISA plate with Parafilm or cellophane and incubate overnight at 4 C. Day 2 (V.F V.G) 3 4 hr F. Protocol: Performing the ELISA 1. 16 20 hr after infecting the insect cells, drain the medium by inverting the plate to remove most of the liquid, and then tapping it on a dry paper towel. 4. Prepare 1X J-RIPA: dilute n x 250 µl 2X J-RIPA in n x 250 µl H 2 O. Add 50 μl of J-RIPA buffer to each well containing insect cells. Shake the plate vigorously (200 400 rev/min) on a microplate shaker for 20 min to lyse the cells. Note: The speed may need to be adjusted depending on the orbital radius of the shaker. It is important to ensure that liquid does not splash out of the wells, and that the entire well bottom remains in contact with the liquid. 5. Meanwhile, prepare the ELISA plate: Wash 4X with 300 µl TBS-T (TBS-Tween; final concentration 0.05% Tween by volume) per well. Let the last wash sit for approximately 5 min. Protocol No. PT3953-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR752243 A Takara Bio Company 8

V. BaculoELISA Titer Kit Protocol continued 6. Aspirate the last wash from the ELISA plate, and immediately transfer the insect cell lysates to the corresponding wells of the ELISA plate. 7. 8. 9. Incubate on a shaker for 30 min (120 240 rev/min). Wash the plate 4X with 300 µl TBS-T per well. Let the last wash sit for approximately 5 minutes. Meanwhile, dilute n x 1.2 µl detection antibody in n x 900 µl TBS-T (e.g., for 12 viruses, dilute 14.4 µl in 10.8 ml TBS-T). 10. Aspirate the last wash from the plate, and add 100 μl of diluted detection antibody to each well. Incubate the plate on a shaker (120 240 rev/min) for 30 min. 11. Wash the plate 4X with 300 µl TBS-T per well. Let the last wash sit for approximately 5 min. 1 1 Meanwhile, dilute the avidin-hrp 1:1,000 (1 µl per ml TBS-T). You will need 900 µl diluted avidin-hrp for each column. Aspirate the last wash from the plate, and add 100 μl of diluted avidin-hrp to each well. Incubate the plate on a shaker (120 240 rev/min) for 30 min. 14. Wash the plate 4X with 300 µl TBS-T per well. Let the last wash sit for approximately 5 min. Attention Note: If you are using a squeeze bottle to dispense buffers, you must be careful to remove all bubbles before adding the TMB substrate by rinsing several times with TBS (without Tween). Bubbles will interfere with the absorbance readings. 15. Meanwhile, mix TMB Substrate Reagents A and B together (1:1; 1.8 ml/column). 16. Add 200 μl of TMB substrate to each well. Incubate the plate on a shaker for 5 min to 1 hr. When a blue color develops, measure the absorbance at 600 700 nm (maximum absorbance at ~650 nm) on a plate reader. Alternatively, stop the reaction with acid (e.g., 25 50 µl of 2N HCl), and measure the absorbance at 430 470 nm (maximum absorbance at ~450 nm) on a plate reader. G. Protocol: Calculating Baculovirus Titers The titer is determined by comparing the volumes of the reference virus (RV) and test virus (TV) that are required to produce the same amount of signal (S; A 650 or A 450 ): Titer (TV) = Titer (RV) x Vol (RV) Vol (TV) The titer of the test virus can be determined most accurately when S is defined as the midpoint, or inflection point, of the curve (Figure 2). The titer of the reference (control) virus is noted on the lot-specific Certificate of Analysis for your BaculoELISA Titer Kit. 5 2 A 655 nm 1.5 1 0.5 Vol (RV) Vol (TV) reference virus test virus 0 16.7µl 5.6 µl 1.9 µl 0.6 µl 0.2 µl 0.06 μl 0.02 μl Volume of virus added to well 0.008 μl Figure Data required to estimate the viral titer of a test virus. The titer of the reference virus (T (RV) ) is known. Volumes of viruses required to produce a signal S (midpoint of the curve; 1.1 in this example) are compared for the reference (Vol (RV) ) and test (Vol (TV) ) viruses, in order to determine the titer of the test virus. Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3953 1 A Takara Bio Company Version No. PR752243 9

V. BaculoELISA Titer Kit Protocol continued You can determine your virus titers manually by plotting the signal versus volume of virus (as shown in Figure 2) or you can download our Microsoft Excel BaculoELISA workbook for convenient, rapid determination of your virus titers. Calculating Baculovirus Titers Electronically 1. Download the Microsoft Excel BaculoELISA workbook from the Online Tools section of our website, www.clontech.com/support/tools.asp. 4. a. b. c. d. Enter the following information into the BaculoELISA workbook: The initial dilution of virus added to the first row of cells (e.g., 3 for the three-fold dilution created by diluting 50 µl virus supernatant with 100 µl medium in Protocol V.B.; see Table I, Row A). The fold dilution between wells ( 3 as the protocol is written; see Table I, Rows B H). The software will calculate the dilution series. The titer of the reference virus. If you are using the reference (control) virus provided with the kit, its titer is noted on the Certificate of Analysis included with the kit. The Certificate of Analysis is also available in the Support section of our website, www.clontech.com/support. The ELISA absorbance (OD) values from the plate reader. The simplest way to enter the absorbance values is to save the data in an Excel-compatible format (e.g., tab- or comma-delimited) within the plate reader software, open the file in Excel, and copy and paste the data into the BaculoELISA workbook. Optionally, you can enter the names of the viruses above the plate readings, and they will be transferred to the table of calculated titers. The software will plot the ELISA signal against the dilution series. Note the background level (from the most dilute row; for example Table I, row H) and the maximum (saturation) signal (from the least dilute row; for example, Table I, row A). Enter the value, S, for the midpoint (inflection point) of each curve. S is typically the steepest part of the curve, where the signal strength is most sensitive to the amount of virus added to the cells. It is necessary in order to compare signals between viruses, and thus to determine the titer of the test viruses. Generally, S is midway between the background level and the saturation level (Figure 2). S = Background + Maximum ExamPLE: If the background is 0.1 and the maximum signal is ~0.6, S 0.35. The software will calculate the titer of each virus in IFU/ml or PFU/ml. 2 Protocol No. PT3953-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR752243 A Takara Bio Company 10

VI. Troubleshooting Table II: Troubleshooting the BaculoELISA Titer Kit Description of Problem No or low signal for all virus dilutions, including the reference virus. The reference virus gives a good signal, but the test viruses do not. Uneven signal curves. Unusually high or low signals are observed for some wells. Explanation A component may have been omitted or added at the wrong concentration. A component has expired. The titers of the test viruses may be too low to measure. Dilutions or washes may have been done incorrectly. Some components may have been omitted from these wells. Some washes may not have been done correctly. Solution Test the Avidin-HRP with the TMB substrate mixture. Check expiration dates. Amplify the test viruses to obtain a higher titer by infecting cells at an MOI of ~0.01 0.1 and harvest the supernatant 72 96 hr post infection. If necessary, refer to the BacPAK Baculovirus Expression System User Manual (PT1260-1) for additional information. Note: The viruses can be amplified in serum-free medium if the insect cells are adapted to serum-free medium; but the viruses should be stored at 4 C, in the dark, in medium containing at least 2% fetal bovine serum (FBS). Repeat the titration. Repeat the titration. Wash with 300 µl of wash buffer for each wash step to ensure that the well is completely filled. VII. References Gershburg, E. et al. (1997) J. Virol. 71(10):7593 7599. Hershberger, A.P. et al. (1994) J. Virol. 68(6):3467 3477. Clontech Laboratories, Inc. www.clontech.com Protocol No. PT3953 1 A Takara Bio Company Version No. PR752243 11